Hepatitis B virus (HBV) is an enveloped dsDNA virus with a ssRNA intermediate form. Correct assembly of the icosahedral core of HBV is required for replication. Reverse transcription of the RNA pre-genome (pgRNA) takes place within the completed capsid, the protein shell of the core. The HBV capsid is constructed from 120 capsid protein (Cp) dimers. In vitro, Cp assembles spontaneously. In vivo, pgRNA is selectively encapsidated in the presence of a great excess of host RNA, even when the Cp is expressed in trans. It is hard to imagine how there can be any selectivity, especially given the +17 charge on the RNAbinding domain of Cp. What prevents misassembly? Based on our in vitro studies, we have developed the hypothesis that HBV assembly is allosterically regulated: we suggest that Cp has assembly-active and - inactive conformations. We have identified different salts and small molecules that favor or inhibit assembly. We have also identified a number of mutations that inhibit and enhance assembly. In parallel, there are high frequency, naturally occurring mutations of the Cp that correlate with unexpected changes in the yield of virions; we hypothesize that these mutations affect assembly proficiency. In order to better understand regulation of HBV assembly, we propose to test our hypotheses that (i) in vitro assembly can be correlated with assembly in vivo and (ii) assembly is subject to allosteric regulation. Assembly of the common naturally occurring mutants will be correlated with phenotype. With assembly-defective Cp, we will describe structural differences between Cp dimers and Cp bound in capsids, a fundamental prediction of allostery. We will investigate how mutations at the dimer interface affect assembly; typically, interfaces between subunits are altered by allosteric transitions. We will examine the RNA-binding and specificity of Cp. Our preliminary data for allostery is strong but requires further investigation. Our results with HBV will advance the question of how assembly is regulated in other viruses. More than 350 million individuals suffer from chronic infection with hepatitis B virus (HBV), including more than 1.25 million Americans. Worldwide, HBV will contribute to 1 million deaths this year. By determining how HBV assembly is regulated, we will characterize a new target for antiviral intervention.